Red/green cyanobacteriochromes acquire isomerization from phycocyanobilin to phycoviolobilin

Abstract Cyanobacteriochromes (CBCRs) are unique cyanobacteria‐specific photoreceptors that share a distant relation with phytochromes. Most CBCRs contain conserved cysteine residues known as canonical Cys, while some CBCRs have additional cysteine residues called second Cys within the DXCF motif, leading to their classification as DXCF CBCRs. They typically undergo a process where they incorporate phycocyanobilin (PCB) and subsequently isomerize it to phycoviolobilin (PVB). Conversely, CBCRs with conserved Trp residues and without the second Cys are called extended red/green (XRG) CBCRs. Typical XRG CBCRs bind PCB without undergoing PCB‐to‐PVB isomerization, displaying red/green reversible photoconversion, and there are also atypical CBCRs that exhibit diverse photoconversions. We discovered novel XRG CBCRs with Cys residue instead of the conserved Trp residue. These novel XRG CBCRs exhibited the ability to isomerize PCB to PVB, displaying green/teal reversible photoconversion. Through sequence‐ and structure‐based comparisons coupled with mutagenesis experiments, we identified three amino acid residues, including the Cys residue, crucial for facilitating PCB‐to‐PVB isomerization. This research expands our understanding of the diversity of XRG CBCRs, highlighting the remarkable molecular plasticity of CBCRs.

Here, we report novel XRG CBCR GAF domains featuring the Cys-Xaa-Asp-Xaa-Xaa-Leu (CXDXXL) motif instead of the conserved WXDXXL motif.These XRG CBCR GAF domains, lacking the DXCF second Cys, exhibited PCB-to-PVB isomerization ability similar to DXCF CBCR GAF domains.They exhibited reversible photoconversion between a Pg dark-adapted state and a teal-absorbing (Pt) photoproduct state but without reversible Cys ligation to the C10 of the chromophore.Through sequence-and structure-based comparisons, we identified three amino acid residues crucial for PCB-to-PVB isomerization activity.This study unravels the diversity of XRG CBCR GAF domains and highlights the remarkable plasticity in the molecular evolution of CBCRs.

| Discovery of novel XRG CBCR GAF domains lacking a conserved tryptophan residue in the WXDXXL motif
CBCRs have expanded their diversity through the acquisition of Cys residues involved in chromophore structural isomerization and/or covalent bond formation with the chromophore during photoconversion (Blain-Hartung et al., 2021;Narikawa et al., 2014;Rockwell et al., 2011Rockwell et al., , 2017;;Rockwell & Lagarias, 2023).We anticipated the discovery of novel CBCRs with unique characteristics due to the acquisition of Cys residues near the chromophore.To explore this possibility, we conducted a search for novel CBCR GAF domains containing Cys residues near the chromophore using protein BLAST.As a result, we identified uncharacterized XRG CBCR GAF domains, which formed a distinct cluster within the XRG lineage (Figure 1b).Among them, four GAF domains, MCC3606390g2 from Microcoleus sp.PH2017_29_MFU_D_A, WP_007354189g2 from multispecies Kamptonema, MBD0393953g from Microcoleus sp.C1-bin4, and WP_106260381g2 from Stenomitos frigidus, have the CXDXXL motif instead of the WXDXXL motif, which is highly conserved in the XRG lineage (Figure 1c, d).In contrast, the other two homologous GAF domains, MBW4492963g from Oscillatoria princeps RMCB-10 and PSB13078g2 from the filamentous cyanobacterium CCP2, retained the WXDXXL motif (Figure 1c, d).

| Spectral properties of novel XRG CBCR GAF domains
The novel XRG CBCR GAF domains were expressed in PCB-producing Escherichia coli and purified using nickel affinity chromatography.SDS-PAGE CBB staining revealed multiple protein bands, with the band around 20 kDa aligning closely with the theoretical molecular sizes of the GAF domains (Figure S2).Notably, only these bands exhibited fluorescence in the presence of zinc ions, indicating that they represent the chromophorylated GAF domains (Figure S2).
MCC3606390g2 exhibited reversible photoconversion between a Pg state peaking at 566 nm and a Pt state peaking at 501 nm (Figure 2a).Similarly, WP_007354189g2 displayed reversible photoconversion between a Pg state peaking at 563 nm and a Pt state peaking at 501 nm (Figure 2b).These Pg-minus-Pt difference spectra were nearly identical (Figure 2c).Acid denaturation analysis indicated that the Pg states were dark-adapted states binding the Z-configured PVB, while the Pt states were photoproduct states binding the E-configured PVB in both cases (Figures 2d and S3A-D).Notably, despite belonging to the XRG lineage and lacking the DXCF second Cys residue (Figure 1b, d), both of these CBCR GAF domains displayed PCB-to-PVB isomerization activity.A relatively small spectral shift 62-65 nm was observed for green/teal photoconversion, suggesting that these molecules do not exhibit reversible Cys ligation such as blue/ green CBCR GAF domains (Ishizuka et al., 2011).To confirm this, iodoacetamide (IAM) was added to both states of MCC3606390g2 (Figure S4).The addition of IAM did not affect either the Pg-to-Pt or Pt-to-Pg photoconversion, leading to the conclusion that the green/teal photocycle occurs without reversible Cys adduct formation.
MBD0393953g and WP_106260381g2 both featuring the same CXDXXL motif, displayed reversible photoconversion between an orange-(Po) or a yellow-absorbing (Py) dark-adapted state (λ max = 574 and 604 nm, respectively) binding the Z-configured PCB, and a Pg photoproduct state (λ max = 553 and 527 nm, respectively) binding the E-configured PCB (Figures 3a, b, e, f and  S3E-H).This contrasts with the results of MCC3606390g2 and WP_007354189g2 (Figures 2, and S3A-D).Notably, these CBCR GAF domains did not demonstrate PCBto-PVB isomerization activity, suggesting that the Cys residue of the CXDXXL motif might not be sufficiently functional in the PCB-to-PVB isomerization process (Figure 3f).It is possible that MCC3606g2 and WP_007354189g2 possess unique amino acid residues crucial for PCB-to-PVB isomerization.
MBW4492963g and PSB13078g2, featuring the canonical WXDXXL motif, demonstrated reversible photoconversion between the Pr dark-adapted states (λ max = 637 and 642 nm, respectively) binding the Z-configured PCB and the Pg photoproduct states (λ max = 532 and 531 nm, respectively) binding the E-configured PCB, which were typical for XRG molecules (Figure 3c-f and Figure S3I-L).

| Identification of amino acid residues crucial for PCB-to-PVB isomerization
To identify amino acid residues crucial for PCB-to-PVB isomerization, we conducted a multiple sequence alignment of six XRG CBCR GAF domains found in this study with typical XRG CBCR GAF domains (Figure 1d).Based on this alignment, we focused on three amino acid residues, Cys, Val, and Ile residues (Cys1015/Val1018/ Ile1076 of the MCC3606390g2) (Figure 1d and Figure 4a).The Cys residue within the CXDXXL motif is conserved among the two PVB-binding molecules (MCC3606390g2 and WP_007354189g2) and the two PCB-binding molecules (MBD0393953g and WP_106260381g2), while the other two PCB-binding molecules (MBW4492963g and PSB13078g2) and typical PCB-binding molecules have Trp residues at this position (Figure 1d).In contrast, the Val and Ile residues are exclusively conserved among the two PVB-binding molecules, whereas the other four PCBbinding molecules have Thr and Ala residues at these positions (Figure 1d).
The triple mutant MCC3606390g2_C1015W_ V1018T_I1076A exhibited photoconversion between a Pr dark-adapted state peaking at 643 nm and a Pg photoproduct state peaking at 529 nm, similar to typical XRG CBCR GAF domains (Figure 4b).No detectable green/teal photoconversion components were observed (Figure 4c).Acid denaturation analysis revealed that the chromophore bound to the triple mutant was not PVB but PCB (Figure 4d).In conclusion, this mutant molecule completely lost its PCB-to-PVB isomerization activity.To investigate the role of each amino acid residue, single and double mutants were constructed and subjected to spectroscopic analysis (Figures 4e-k, S2, S5, and S6).These mutants displayed heterogeneous binding behaviors to both PVB and PCB, as indicated by the absorption spectra of the native and denatured samples (Figures 4e-k, S5, and S6).To accurately evaluate these heterogeneous samples, we established a light illumination protocol to photoconvert one of the PVB-and PCB-binding components specifically (see the "Materials and Methods" section in detail).This protocol generated three preparations:  (Fushimi et al., 2016) and PVB-binding ones (AM1_6305g1, graysolid) (Hasegawa et al., 2018).
(A) PVB-and PCB-binding Z-isomers, (B) PVBbinding Z-isomer and PCB-binding E-isomer, and (C) PVB-and PCB-binding E-isomers (Figure S7).The absorption spectra of preparations A-to-C were summarized in Figure 4.The Z-minus-E difference absorption spectra of the PVB-binding component (A-minus-B) and the PCB-binding component (Bminus-C) were summarized in Figure S6.We first To quantitatively assess the PCB-to-PVB isomerization activity, we normalized the spectra of preparation A, where both the PVB-and PCB-binding components were present as dark-adapted states (Figure S7), by the peak absorbance of the Pg dark-adapted states of the PVBbinding components (Figure 4l).However, some mutations affect the absorption shape of the PCB-binding component, preventing quantitative evaluation.Instead, we calculated the ratio of PVB to PCB in the variant molecules from the spectra of acid-denatured preparations as previously reported (Figure S5 and Table 1) (Ma et al., 2012).The amount of the PCB-binding component in the wild-type was estimated as 1.8%, indicating almost full PCB-to-PVB isomerization activity.Among the single mutants, the C1015W mutant contained the largest amount of the PCB-binding component (14.7%) compared to the V1018T and I1076A mutants (6.9% and 0.6%, respectively).Consistently, the double mutants containing the C1015W mutation (C1015W_V1018T, C1015W_I1076A) contained a greater number of PCBbinding components (45.5% and 33.9%, respectively) than the V1018T_I1076A double mutant (7.4%).C1015W mutation had the most significant inhibitory effect on the PCB-to-PVB isomerization activity.Furthermore, for both the single and double mutants, V1018T mutation (6.9% and 45.5% for the V1018T single and C1015W_V1018T double mutants, respectively) showed more significant inhibitory effect on the PCB-to-PVB isomerization activity than I1076A mutation (0.6% and 33.9% for the I1076A single and C1015W_ I1076A double mutants, respectively).Not simple additive effects but synergetic effects were observed for the double and triple mutants except for the V1018T_I1076A double mutant (7.4%).In conclusion, Cys1015 appears to be the most crucial, while Val1018 and Ile1076 seem to play supportive roles with higher contribution of Val1018.

| Introduction of key amino acid residues into other XRG CBCR GAF domains
The three amino acid residues responsible for PCBto-PVB isomerization ability in MCC3606390g2 were introduced into the other XRG CBCR GAF domains, WP_106260381g2 and AnPixJg2, to determine if these three residues alone are sufficient for the PCB-to-PVB isomerization (Figure 1d).However, the purified WP_10620381g2_T566V_A624I and AnPixJg2_W289C_T292V_A350I mutant molecules did not exhibit zinc-induced fluorescence in the SDS-PAGE gel or specific absorbance in the visible light region, except for a faint heme-like absorption, likely due to the contaminant protein from E. coli (Figures S2 and S8).In conclusion, these mutants failed to bind any chromophore, preventing the evaluation of the effect of these amino acid residues on PCB-to-PVB isomerization activity.

T A B L E 1
The ratio of PVB and PCB incorporated into MCC3606390g2 variant molecules.
Despite belonging to the XRG lineage, MCC3606390g2 and WP_007354189g2 bound PVB instead of PCB, suggesting that these two CBCR GAF domains acquired PCB-to-PVB isomerization ability through alterations in their amino acid residues during the molecular evolution process (Figures 1 and 2).Site-directed mutagenesis revealed that replacing three amino acid residues (C1015W, V1018T, and I1076A) with those from MBW4492963g and PSB13078g2 led to a complete loss of PCB-to-PVB isomerization ability (Figure 4b-d).
To explore the contribution of these three amino acid residues to PCB-to-PVB isomerization activity, we examined the corresponding residues of the Pr darkadapted state structure of AnPixJg2 (Figure 5a) (Narikawa et al., 2013).Site-directed mutagenesis revealed that replacing Cys1015 with Trp had the most significant inhibitory effect on PCB-to-PVB isomerization (Figure 4).In the Pr dark-adapted state of AnPixJg2, the corresponding Trp residue forms a π-π stacking with the D-ring and a hydrogen bond with the A-ring nitrogen, crucial for maintaining a stable chromophore conformation to absorb red light (Figures 5a  and S1C).However, the Cys residue would lack both interactions.Consequently, the A-ring, not stabilized by the hydrogen bond, may be twisted relative to the B-C plane of the rings (Figure 5b, c).Homologous molecules, MBD0393953g and WP_106260381g2 molecules possessing the same CXDXXL motif did not exhibit PCBto-PVB isomerization ability, likely due to the influence of the other two residues (Figures 1 and 3f).The darkadapted states of these molecules absorbed in the yellow-to-orange region, shorter than the red absorption of typical red/green reversible molecules like AnPixJg2 (Figure 3a, b).This blue-shifted feature would result from the A-ring twist due to the absence of the Trp residue.
MCC3606390g2 features a Val1018 residue near the A-ring (Figure 5a), which, in addition to Cys1015, plays a crucial role in PCB-to-PVB isomerization (Figure 4).Val1018 likely contributes to stabilizing the twisted A-ring through hydrophobic interaction to a greater extent.In vitro reconstitution analysis with the PCB chromophore has shown that the typical DXCF CBCR GAF domain, TePixJg, binds PCB immediately after reconstitution and undergoes blue/green reversible photoconversion (Ishizuka et al., 2011).TePixJg demonstrates both PCB-to-PVB isomerization activity and reversible Cys ligation, where the second Cys residue binds to the C10 of the chromophore in the dark-adapted state of Pb to absorb blue light.In this context, the blue light absorption in the dark-adapted state incorporating the PCB chromophore immediately after reconstitution is likely due to Cys adduct formation between C10 of PCB and the second Cys residue.On the other hand, green light absorption in the photoproduct state, even after incorporating the PCB chromophore immediately after reconstitution, may be attributed to the high distortion of the A-ring.This highly distorted A-ring is believed to be a trigger for PCB-to-PVB isomerization (Fushimi & Narikawa, 2021b).A similar A-ring twist, facilitated by Cys1015 and Val1018, is presumed to also trigger PCBto-PVB isomerization in the MCC3606390g2 molecule.
Ile1076, corresponding to the Ala residue in AnPixJg2, is situated at the β-face of the D-ring, opposite to the Cys/Trp residue (Figure 5a).The bulky and hydrophobic side chain of Ile1076 contributes to stabilizing the D-ring conformation through hydrophobic interaction, complementing the role of the Trp residue on the opposite side.In typical DXCF CBCR GAF domains, PCBto-PVB isomerization occurs under dark conditions, with the second Cys residue ligating to C10 of the chromophore (Ishizuka et al., 2011).In this context, the high distortion of the C4 = C5 double bond is established by the dual fixation of the chromophore at the A-ring and C10 position via the first and second Cys residues, respectively.This highly unstable and rigid conformation would trigger reduction at the C4 = C5 double bond and concomitant oxidation of the C2-C3 single bond of the A-ring (Fushimi & Narikawa, 2021b;Rockwell, Martin, Gulevich, & Lagarias, 2012).As the MCC3606390g2 molecule did not exhibit reversible Cys adduct formation with the C10 of the chromophore, an alternative method must establish the high distortion of the C4 = C5 double bond.We recently reported that the DXCF CBCR molecule AM1_6305g1 did not require the second Cys residue for PCB-to-PVB isomerization (Fushimi & Narikawa, 2021b).Replacement of the second Cys with Ser did not affect the isomerization activity.In this molecule, amino acid residues near the D-ring would complement the role of the second Cys by firmly fixing the D-ring but not the C10 of the chromophore.Ile1076 in MCC3606390g2 would similarly contribute to the high distortion of the C4 = C5 double bond by fixing the D-ring.
Although highly twisted A-ring has been observed for the Pg photoproduct state of the red/green CBCR GAF domains (Lim et al., 2018;Xu et al., 2020), these domains do not show PCB-to-PVB isomerization activity at all (Chen et al., 2012;Narikawa et al., 2008;Rockwell, Martin, & Lagarias, 2012c).Because the dark-adapted state but not the photoproduct state should be first generated when the holoprotein has been folded, PCB-to-PVB isomerization within the CBCR GAF domains would occur in the dark-adapted states.In this context, highly twisted A-ring in the Pg photoproduct state would not result in PCB-to-PVB isomerization.
Among the different MCC3606390g2 mutant molecules, the C1015W and C1015W_V1018T mutants with Trp and Ile residues at the Cys1015 and Ile1076 positions, respectively, exhibited a broad orange-to-red absorption in the dark-adapted states for the PCB-binding components, whereas the other mutants displayed sharp red absorption (Figure 4e-k).Bulky residues (Trp and Ile) on both the αand β-faces of the D-ring could create a confined environment around the D-ring, potentially resulting in broad absorption.
The V1018T_I1076A double mutant of MCC3606390g2 shares the same combination of amino acid residues as the homologous proteins MBD0393953g and WP106260381g2, which are crucial for PCB-to-PVB isomerization (Figure 1d).Notably, the V1018T_I1076A double mutant retained partial PCB-to-PVB isomerization activity, whereas the wild-type molecules MBD0393953g and WP10620381g2 did not exhibit detectable isomerization activity.This suggests that MCC3606390g2 may possess additional unidentified amino acid residue(s) crucial for PCB-to-PVB isomerization activity (Figures 3a,b,f,4k,and S6G,N).Further comparisons based on sequence and structure would reveal additional residues in the future.
A previous study identified Lyn8106_0097 and Lyn.aest_0230, referred to as RB2 with CXDXXL motifs, as equivalent to MCC3606390g2 and WP_007354189g2.These RB2 molecules bind PCB and undergo red/blue photocycles, indicating the formation of a covalent bond to the C10 of the chromophore during photoconversion through the Cys residue within the motif (Blain-Hartung et al., 2021).While the Cys residue at the same position reversibly binds to the C10 of the chromophore in the RB2 molecules, it contributes to PCB-to-PVB isomerization in MCC3606390g2 and WP_007354189g2.RB2 molecules belong to the NpR3784 lineage rather than the XRG lineage (Figure 1a).This NpR3784 lineage, like the XRG lineage, lacks the second Cys residue within the DXCF motif, and its typical molecules exhibit a red/green photocycle similar to XRG CBCR molecules.The typical molecules of the NpR3784 lineage do not have a Trp residue at the Cys position within the CXDXXL motif.Instead, the Val residue is highly conserved among the NpR3784 lineage, and the interaction network via this residue is likely different from that via the Trp residue in the XRG lineage, potentially resulting in different functionality of the Cys residue at the same position.
The PCB-binding MCC3606390g2 triple mutant molecule undergoes red/green reversible photoconversion with a spectral shift of over 100 nm, while the PVBbinding MCC3606390g2 wild-type molecule undergoes green/teal reversible photoconversion with only a 65 nm spectral shift (Figures 2a and 4b).This difference likely arises from differences in the chromophore structure.The PCB chromophore has four tetrapyrrole rings that are fully conjugated, whereas the A-ring of the PVB chromophore is deconjugated from the other rings (Figure S1A).In typical PCB-binding XRG molecules, both rings A and D exhibit high levels of twisting relative to the rings B-C plane in the Pg state compared to the Pr state (Lim et al., 2018 ;Xu et al., 2020), resulting in a significant blue shift.However, PVB incorporation cancels the effect of A-ring distortion on absorption, leading to a smaller spectral shift.

| Protein expression and purification
The proteins were expressed in E. coli C41 pKT271_C0185, which was incubated at 37 C until the OD 600 reached a range of 0.4-0.8.Isopropyl-β-Dthiogalactopyranoside was then added to induce expression at 18 C.The cells were collected by centrifugation at 5000Â g for 20 min and frozen at À80 C for 30 min or longer.Afterward, the cells were suspended in a lysis buffer (20 mM HEPES-NaOH pH 7.5, 0.1 M NaCl, and 10% (w/v) glycerol) and disrupted using an Emulsiflex C5 high-pressure homogenizer at 12,000 psi (Avestin, Inc., Ottawa, Canada, ON, Canada).The mixtures were centrifuged at 17,000Â g for 60 min to separate the pellets and supernatants.The collected supernatants were filtered through a 0.8-μm cellulose acetate membrane and loaded onto a nickel-affinity His-trap column (GE Healthcare, Piscataway, NJ, USA) using ÄKTA pure (GE Healthcare, Piscataway, NJ, USA).His-tagged proteins were purified using a lysis buffer containing 100-400 mM imidazole with a linear gradient system (1 mL/ min, 15 min) after the column was washed using a lysis buffer containing 100 mM imidazole.EDTA (final concentration, 1 mM) was added to the purified protein, which was incubated on ice for 1 h and then dialyzed against the lysis buffer to remove imidazole and EDTA.

| Spectral analysis
UV-2600 spectrophotometer (SHIMADZU, Kyoto, Japan) was used to record the ultraviolet and visible absorption spectra of the native proteins.An Opto-Spectrum Generator (Hamamatsu Photonics, Inc., Hamamatsu, Japan) was utilized to produce monochromic light for photoconversion.Denaturation of proteins was achieved using 1 M HCl/8 M urea and the absorption spectra of the denatured proteins were measured at room temperature.The Z-configured PCB-and PVB-binding components of the MCC3606390g2 mutants were generated by irradiation with 500 nm light for 1 min (Figure S7A).A mixture of the E-configured PCB-binding component and the Zconfigured PVB-binding component of the MCC3606390g2 mutants was obtained by successive irradiation with 500 nm light for 1 min and 650 nm light for 1 min (Figure S7B).Additionally, a mixture of Econfigured PCB-and PVB-binding components of the MCC3606390g2 mutants was generated by successive irradiation with 590-nm light for 1 min and 650-nm light for 1 min (Figure S7C).The ratio of PCB and PVB bound to the MCC3606390g2 mutants were estimated from absorbances at 592 nm derived from Z-PVB and 662 nm derived from Z-PCB of these denatured absorption spectra (Figure S5), based on the calculation method presented in the previous study (Ma et al., 2012).

| Electrophoresis
The purified proteins were diluted in a buffer (60 mM dithiothreitol (DTT), 2% (w/v) sodium dodecyl sulfate (SDS), and 60 mM Tris-HCl pH 8.0) for SDS-PAGE.Following denaturation by heat shock at 95 C for 3 min, the samples were electrophoresed at room temperature using a 12% (w/v) acrylamide gel.Subsequently, the electrophoresed gels were immersed in 20 mM zinc acetate at room temperature for 30 min to visualize the fluorescence of the purified proteins.Blue light (λ max = 470 nm) and green light (λ max = 527 nm) were used for illumination, and a WSE-5500 VariRays (ATTO, Tokyo, Japan) equipped with a short path filter (passing through <562 nm) and a long path filter (passing through >600 nm) was utilized to capture the fluorescence bands.The fluorescence bands were then imaged using a WSE109 6100 LuminoGraph (ATTO, Tokyo, Japan).Subsequently, the gels were stained with Coomassie brilliant blue R-250 (CBB) after observation.

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I G U R E 1 Legend on next page.HOSHINO ET AL.
Phylogenetic analysis, domain architecture, and multiple sequence alignment of novel XRG CBCRs.(a) Phylogenetic tree depicting representative CBCR GAF domains.XRG CBCRs are highlighted in red.(b) Phylogenetic tree specifically focusing on XRG CBCR GAF domains.Novel XRG CBCR GAF domains from this study are denoted in pink (MCC3606390g2 and WP_007354189g2), purple (MBD0393953g and WP_106260381g2), and blue (MBW4492963g and PSB13078g2).(c) Domain architecture of novel CBCRs, with GAF domains colored based on the absorbed light colors in the dark-adapted state and photoproduct state.(d) Multiple sequence alignment of XRG CBCR GAF domains, with the first amino acid number listed on the left.Conserved amino acid residues are highlighted in bold and the three key amino acid residues are colored in red.

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I G U R E 2 Absorption spectra of the novel PVB-binding XRG CBCR GAF domains.(a) Absorption spectra of native MCC3606390g2.(b) Absorption spectra of native WP_007354189g2.(c) Normalized difference spectra of native MCC3606390g2 (orange) and WP_007354189g2 (salmon pink).(d) Normalized difference spectra of denatured MCC36063390g2 (orange) and WP_007354189g2 (salmon pink) with reference samples of PCB-binding (AM1_C0023g2, gray-dash)

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I G U R E 5 Amino acid residues crucial for the PCB-to-PVB isomerization in MCC3606390g2.(a) The three residue positions highlighted on the structure of the Pr dark-adapted state of AnPixJg2 (PDB ID: 3W2Z).(b) Conformation of PCB incorporated into AnPixJg2 scaffold having WXDXXL motif.(c) Predicted conformations of PCB or PVB incorporated into MCC3606390g2 scaffold having CXDXXL motif.The dashed line indicates the hydrogen bond between the side chain of Trp of the WXDXXL motif and the hydroxyl group of the A-ring of PCB.